The pyruvate dehydrogenase complex (PDC) plays a pivotal role in the metabolism of pyruvate for energy production and for biosynthetic processes. Genetic defects of the human PDC are associated with congenital lactic acidosis, variable neurological disability, and often early death. The long term goals of this project are to investigate the molecular basis of genetic defects of PDC in affected patients and to enhance our understanding of the structure-function relationships and regulation of the pyruvate dehydrogenase (E1) component of PDC, both at the level of protein and DNA. During the past four years, we and others have identified specific mutations in the coding region of the human E1alpha gene localized on chromosome X. However, little is known about the functional significance of these mutations at the protein level. Furthermore, our understanding of the clinical heterogeneity of E1 deficiency remains fragmentary. The E1 component, an alpha2beta2 tetramer, catalyzes the rate-limiting step in the complex and is subject to regulation by covalent modification; our understanding of the nature of its active site and the roles played by these two subunits is very limited. Although the E1 (and hence PDC) is subject to short-term regulation, its long-term regulation at the transcriptional level by hormones is poorly understood. Initial analyses of the promoter regions of the human E1alpha and E1beta genes indicate that these promoters may represent unique variations to transcriptional regulation of "housekeeping genes." Five specific aims proposed in this renewal application are to (i) investigate the roles of specific amino acid residues in the active site, (ii) investigate the structure-function relationships of the two subunits and the roles of the three phosphorylation sites in regulation of catalytic activity, (iii) analyze the transcriptional regulation of the E1alpha promoter region, (iv) analyze the transcriptional regulation of the human E1beta promoter- regulatory region, and (v) characterize genetic defects in PDC-deficient patients. We will overexpress site-directed mutant human E1 proteins to investigate the basis for structure-function relationships of E1. Kinetic parameters, spectral and binding properties of the mutant proteins will be determined. Transcriptional activity of the wild-type and mutant promoters will be analyzed using DNase I footprinting, gel mobility shift and expression of a reporter gene in transfected cells. Genetic defects will be analyzed using enzyme assays, Western and Northern analyses, and sequencing of patient-specific cDNAs. Selected mutations will be recreated using site-directed mutagenesis and overexpression of the mutant proteins for functional analysis. Our multifaceted experimental approach is designed to enhance our understanding of the structure-function relationships of E1 in catalysis, transcriptional regulation of the E1 genes, and the molecular basis of genetic defects of E1.